Ratiometric determination of glycated protein

ABSTRACT

The present invention provides methods for determining a ratio of an amount of a glycated form of a protein to a total amount of the protein in a sample containing the glycated protein, the glycosylated protein, or the glycoprotein. The method incorporates lateral flow test strip or vertical flow test strip devices having negatively charged carboxyl or carboxylate groups and hydroxyboryl groups immobilized and interspersed on a solid support matrix. The solid support matrix may include derivatives of cellulose (e.g., carboxy cellulose) derivatized with carboxylic acid (e.g., carboxylate, carboxyl) groups and hydroxyboryl compounds including phenylboronic acid (e.g., phenylborate), aminophenylboronic acid, boric acid (e.g., borate), or other boronic acid (e.g., boronate) compounds. The present invention is usefi.il for monitoring glycation or glycosylation of hemoglobin or albumin for monitoring glycemic control (e.g., glycemia in diabetes).

PRIORITY INFORMATION

This application claims priority to U.S. application Ser. No.60/265,229, filed Jan. 31, 2001.

FIELD OF THE INVENTION

This invention relates to a method for the quantitation of percentglycated protein in a biological sample suitable for use with areflectance meter such as used in the self monitoring of blood glucoseconcentration by diabetics.

BACKGROUND OF THE INVENTION

Control of blood glucose concentrations in diabetics has been shown todecrease the frequency and severity of long-term microvascular andneurologic complications of the disease. The measurement of glycatedhemoglobin and protein in blood are used to determine how well bloodglucose concentration has been managed over an extended time period.

The rate of formation of glycated hemoglobin is directly related to theglucose concentration in blood.

The average red blood cell life span is 120 days, so quantitation of thepercent glycation of hemoglobin has been correlated to a measure of theaverage glucose concentration over the previous 2 to 3 months which is ameasure of glycemic control over that time period (see “Diabetes Controland Complications Trial Research Group, The effect of intensivetreatment of diabetes on the development of progression of long-termcomplications in insulin-dependent diabetes mellitus”, New EnglandJournal of Medicine, 329, 977–986 (1993), and “American DiabetesAssociation, Tests of Glycemia in Diabetes”, Diabetes Care, 20 (suppl.1), S18–S20 (1997)).

Glucose also attaches to non-hemoglobin proteins in blood, for examplealbumin. Since albumin is the most abundant serum protein and itscirculating half-life is about 20 days, the concentration of glycatedprotein is a measure of the average glucose concentration over theprevious 2 to 3 weeks. The measure of glucose directly gives the glucoseconcentration at the time of measurement.

An immobilized dihydroxyboryl compound has been reported as useful tobind to the 1,2 cis diols of the carbohydrate of glycated proteins toseparate them from non-glycated proteins. Use of this technology in acolumn chromatography method to determine percent glycation has beenreported (see, U.S. Pat. No. 4,269,605 issued May 26, 1981 to Dean andU.S. Pat. No. 5,284,777, issued Feb. 8, 1994 to Rosenthal). Thesemethods are said to use the boronate derivative immobilized onto agarosebeads in a column to separate glycated from non-glycated proteins in thesample. These methods require specific dilutions and pipettings of thesample so as to not overload the capacity of the affinity binder affixedto the agarose beads. The use of a boronate derivative immobilized onagarose beads would not appear to lend itself to a strip application.

U.S. Pat. No. 5,110,745 issued May 5, 1992 to Kricka, et al., is said todescribe methods of detecting glycated protein in a sample wherein thesample is contacted with a defined excess of a boronate compound insolution. The resulting unbound boronate is said to be measured bybinding it to an immobilized glycated molecule on a support matrix andmeasuring the amount of glycated molecule left un-complexed. This methodappears to require a number of steps including, a separate reaction insolution before application to a solid support, dilution of the sampleto assure that the amount of binder added to the biological sample is inexcess, performance of a separate assay to determine the percentage ofprotein glycation and multiple binding and washing steps.

A dipstick method for the measurement of glycated hemoglobin is said tobe described in U.S. Pat. No. 4,861,728 issued Aug. 29, 1989 to Wagner.This method is said to involve contacting of a hemoglobin binding agentlinked to a solid support with a lysed blood sample previously mixedwith a dihydroxyboryl compound linked to a fluorescent label. Thesupport is said to bind non-glycated hemoglobin and fluorescent labeledglycated hemoglobin. The fluorescent label is said to bind to glycatedhemoglobin through the dihydroxyboronyl compound. The solid phase isremoved from the sample and total hemoglobin is measured by reflectancephotometry while glycated hemoglobin is measured using fluorescence.This method is said to require an addition of an amount of fluorescentlabeled dihydroxyboryl reagent to the sample and a rinsing step after itis removed from the sample. Further it requires two differentmeasurement methods for the quantitation.

In other assays, (see, e.g., Japanese Pat. No. 6,058,936, European Pat.No. 455225, and published PCT application WO 96/03657) a boronatederivative coupled to a detectable label (such as a fluorescentcompound, a chemiluminescent compound, isotope, enzyme or other label)is said to be used. Both the glycated and non-glycated proteins arebound to a solid support using a general affinity binder such as anantibody. The boronate-label complex is added and the amount of labelthat remains bound to the solid support is measured. Each of these typesof methods requires the additional step of labeling the glycatedprotein. In addition, these assays use different measurement methods toquantitate total and glycated proteins.

Published PCT application WO 9840750 (published Sep. 17, 1998) is saidto describe a method of determining the percentage of glycatedhemoglobin in which immobilized boronate binds glycated hemoglobin inthe sample. The amount of glycated hemoglobin bound is said to beproportional to the fraction of glycated hemoglobin in the sample. Thisis said to eliminate the need for measuring nonglycated hemoglobin todetermine the percent glycation. However, it appears the method resultsmay vary depending on the incubation time of glycated protein with theboronated support.

Consequently, there is a need for a simple, fast and efficient method toquantitate the amount of glycated protein in a biological sample thatdoes not require dilution of the sample, requires minimal proceduralsteps, may be utilized in conjunction with a simple detection devicesuch as a hand held reflectance meter, and is adaptable to a standardstrip assay.

SUMMARY OF THE INVENTION

The present invention is directed to methods for the quantitation ofglycated proteins in a biological sample, devices utilizing thesemethods and kits comprising these devices.

The results from measurement of glucose, glycated protein and glycatedhemoglobin may provide a more complete picture of glycemic control.Measurement of immediate glucose concentration may be used foradjustment of medications, diet or exercise. For measurement of mediumterm glycemic control, measurement of glycated albumin concentration mayallow one to follow effects of recent changes in lifestyle. Formeasurement of long term glycemic control, measurement of glycatedhemoglobin concentration may allow one to monitor overall effects ofchanges. It would be convenient to have a test for all three in a formatthat could be used in a doctor's office lab so results could bediscussed with the patient during the visit or, alternatively, used bythe patient at home. Currently, blood glucose is routinely monitoredusing hand held meters and easy to use strips. Unfortunately, similartests for glycated proteins and hemoglobin have not been available.

The methods of the present invention provide methods of quantitatingglycated protein in a biological sample. The biological sample iscontacted with a solid support matrix under conditions where bothglycated and non-glycated protein are bound to the solid support matrix.An amount of a first buffer is added sufficient to rinse off unboundprotein. A first bound protein measurement is made to determine total(glycated and non-glycated) bound protein. A second buffer is added tothe solid support matrix which changes the conditions so that glycatedprotein is bound and non-glycated protein is not substantially bound andis added in an amount sufficient to rinse off unbound (non-glycated)protein. A second bound protein measurement is made. Glycated protein isquantitated using the first and second bound protein measurements.

According to one aspect, the present invention is directed to a methodof quantitation of glycated protein in a sample which comprises: (a)contacting a solid support matrix which comprises a negatively chargedgroup and a hydroxyboryl compound and which has a measurement area, withan aliquot of biological sample sufficient to cover said measurementarea; (b) contacting said solid support matrix with an aliquot of afirst buffer sufficient to rinse off unbound protein, wherein said firstbuffer has a pH selected to allow both glycated and non-glycated proteinto be bound to said solid support matrix;(c) quantitating protein boundto said measurement area using measurement of a selected property ofsaid protein to give a first bound protein reading; (d) contacting saidsolid support matrix with an aliquot of a second buffer sufficient torinse off unbound protein, wherein said second buffer has a pH selectedto allow glycated protein to be bound to said solid support matrix butwhere non-glycated protein is not substantially bound to said solidsupport matrix; (e) quantitating protein bound to said measurement areausing measurement of the property measured in step (c) to give a secondbound protein reading; and (f) calculating percentage of glycatedprotein using said first and second bound protein readings.

According to an alternate aspect, the present invention is directed to amethod for quantitation of amount of glycated protein in a biologicalsample which compromises: (a) contacting a solid support matrix whichcomprises a negatively charged group and a hydroxyboryl compound andwhich has a measurement area with an aliquot of a biological samplesufficient to cover said measurement area; (b) contacting said solidsupport matrix with an aliquot of a first buffer sufficient to rinse offunbound protein, wherein said first buffer has a pH of about 5.0 toabout 7.0; (c) quantitating protein bound to said measurement area togive a first bound protein reading; (d) contacting said solid supportmatrix with an aliquot of second buffer sufficient to rinse off unboundprotein, wherein said buffer has a pH of about 8.0 to about 10.0; (e)quantitating protein bound to said measurement area to give a secondbound protein reading; and (f) calculating percentage of glycatedprotein in said sample using said first bound protein reading and saidsecond bound protein reading.

Preferably, and for convenience, the first and second bound proteinreadings measure the same property. Preferably, the property measured isan optical reading. More preferably, the optical reading is absorbanceor reflectance at a specified wavelength.

According to one preferred aspect of the present invention, a method forquantitation of glycated hemoglobin in a biological sample is providedwhich comprises: (a) bringing the biological sample into contact with asolid support matrix which comprises negatively charged groups and adihydroxyboryl compound and which has a measurement area, at a sampleapplication site which is in communication with the solid supportmatrix; (b) adding an aliquot of a first buffer at the sampleapplication site wherein the first buffer has a pH of about 5.0 to about7.0; (c) making a first optical reading of said measurement area at awavelength at which hemoglobin absorbs light; (d) adding an aliquot of asecond buffer at the sample application site wherein the second bufferhas a pH of about 8.0 to about 10.0; (e) making a second optical readingof the measurement area at a wavelength at which hemoglobin absorbslight; and (f) calculating the percentage of glycated hemoglobin in theblood sample using the first and second optical readings. Where thebiological sample is a blood sample comprising red blood cells, thesample is contacted with a red blood cell lysing agent before the firstoptical reading is made. The blood sample may be pre-treated with a redblood cell lysing agent prior to being brought into contact of the solidsupport. Alternatively, the first buffer may further comprise a redblood cell lysing agent or the solid support matrix may be treated witha red blood cell lysing agent before it comes in contact with thebiological sample.

According to another preferred aspect of the present invention, a methodfor quantitation of a glycated non-hemoglobin protein is provided whichcomprises: (a) bringing the biological sample into contact with a sampleapplication site which is in communication with a solid support matrixwhich comprises negatively charged groups and a dihydroxyboryl compoundand which has a measurement area; (b) adding an aliquot of a firstbuffer to the sample application site wherein the first buffer has a pHbetween about 5.0 and about 7.0; (c) making a first optical reading ofthe measurement area at a wavelength at which the protein absorbs light;(d) adding an aliquot of a second buffer to the sample application sitewherein the second buffer has a pH between about 8.0 and about 10.0; (e)making a second optical reading of the sample application site at awavelength at which the protein absorbs light; and (f) calculating thepercentage of glycated protein in the biological sample using the firstand second optical readings. Suitable biological samples for use in themethod of this aspect of the present invention include plasma and serumsamples. A suitable glycated protein for quantitation according to thisaspect is albumin. For the quantitation of certain glycated proteins itmay be preferred that the protein be labeled with a suitable proteinspecific labeling agent.

Preferred buffer systems for use as the first buffer include MES[2(N-morpholino) ethanesulfonic acid], MOPS[3(N-morpholino)propanesulfonic acid] and HEPES[N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid].

Preferred buffers for use as the second buffer include ammonium acetateand taurine buffers.

According to another aspect of the present invention, a diagnosticdevice for quantitation of glycated protein utilizing the methods aboveis provided. Preferably the protein is hemoglobin or albumin.

In another aspect, a kit is provided which comprises the diagnosticdevice described above; a first buffer having a pH of about 5.0 to about7.0; and a second buffer having a pH of about 8.0 to about 10.0.

Definitions

In accordance with the present invention and as used herein, thefollowing terms are defined to have the following meanings, unlessexplicitly stated otherwise:

The term “alkyl” refers to saturated aliphatic groups includingstraight-chain, branched-chain and cyclic (including polycyclic) groups.

The term “carboxylate” or “carboxy” refers to the group —COOH.

The term “phosphate” refers to the group —PO₄.

The term “sulfate” refers to the group —SO₄.

The term “sulfinate” refers to the group —SO₂H.

The term “sulfonyl” refers to the group —SO₃H.

“Hb” refers to hemoglobin.

“HEPES” refers to [N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid].

“K”, when used in the context of measurement of amount of light absorbedand reflectance, refers to the absorption coefficient.

“MES” refers to [2(N-morpholino)ethanesulfonic acid].

“MOPS” refers to [3-(N-morpholino)propanesulfonic acid].

“S”, when used in the context of measurement of amount of light absorbedand reflected, refers to the scattering coefficient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph of the absorbance of glycated hemoglobin (closedtriangles) and total hemoglobin (open triangles) bound to the solidsupport in the assay and a graph of the ratio of glycated to totalhemoglobin (open circles) versus soak time. Different supports wereused, having been derivatized with boronate for increasing lengths oftime.

FIG. 2 depicts a graph of optical absorbance of bound hemoglobin at 415nm vs. incubation time for blood lysate of a diabetic sample comparing asingle measurement assay (“

” for glycated) to the method of the present invention (circles fortotal Hb, open triangles for glycated Hb).

FIG. 3 depicts a graph of optical absorbance at 415 nm of glycatedhemoglobin or fraction of glycated hemoglobin vs. incubation time forblood lysate of a diabetic sample comparing a single measurement assay(adapting the method of published PCT application WO 98/40750 “

”) to the method of the present invention (open triangles).

FIG. 4 depicts a graph of the absorbance of hemoglobin binding to thecarboxy cellulose support (no added boronate groups) over a pH range.

FIG. 5 depicts a graph of the absorbances of hemoglobin (closed circles)and of albumin (open triangles) bound to the boronated support over a pHrange.

FIGS. 6A, 6B and 6C depict three strip configurations which may be usedaccording to the methods of the present invention.

FIG. 7 depicts a graph of K/S vs. percent glycated hemoglobin (“

”) showing proportionality of present invention assay results to theconcentration of glycated hemoglobin in the sample in a strip assayformat using the method of the present invention (open triangles depicttotal hemoglobin).

FIG. 8 depicts a graph of K/S fraction of glycated hemoglobin (glycatedhemoglobin divided by total hemoglobin) vs. percent glycated hemoglobinshowing proportionality of the fraction to glycated hemoglobinconcentration in the sample in a strip assay format using the method ofthe present invention.

FIG. 9 depicts a graph of optical absorbance at 280 nm for total (opentriangles) and glycated albumin (“

”) vs. relative fructosamine content (proportional to fractional amountof glycated albumin) in a sample.

FIG. 10 depicts a graph of the ratio of glycated albumin to totalalbumin (absorbance at 280 nm) vs. relative fructosamine content(proportional to the fractional amount of glycated albumin) in a sample.

FIG. 11 depicts a graph of optical absorbance at 415 nm of bound totalhemoglobin (closed triangles) and glycated hemoglobin (open triangles)vs. whole blood sample volume in a lateral flow device. (See FIG. 6A)

FIG. 12 depicts a graph of the ratio of glycated hemoglobin divided bytotal hemoglobin vs. whole blood sample volume in a lateral flow device.(See FIG. 6A)

FIG. 13 depicts a graph of optical absorbance at 415 nm of totalhemoglobin (open triangles) and glycated hemoglobin (“

”) vs. dilution of a normal blood sample lysate.

FIG. 14 depicts a graph of optical absorbance at 415 nm of totalhemoglobin (open triangles) and glycated hemoglobin (“

”) vs. dilution of a diabetic blood sample lysate.

FIG. 15 depicts a graph of optical absorbance at 415 nm of glycatedhemoglobin divided by total hemoglobin vs. dilution of a normal (“

”) and diabetic blood (open triangles) sample lysate.

FIGS. 16A and 16B depict graphs showing correlation between whole bloodassay results using an assay of the present invention and two othercommercially available assays for hemoglobin A1c. FIG. 16A depicts aplot of results obtained using an assay of the present invention (SeeExample 9) versus results obtained using the Roche® Integra 400 (RocheDiagnostics). FIG. 16B depicts a plot of results obtained using an assayof the present invention (See Example 9) versus results obtained usingthe DCA 2000 (Bayer).

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect, the present invention provides a method forquantitation of glycated proteins in a biological sample in which thesample and reagent(s) are applied to a solid support matrix whichpreferably comprises a negatively charged group and a dihydroxyborylcompound. Measurement of the total and glycated proteins is done at asingle location on the solid support. This measurement may convenientlybe done by measuring a selected property of the protein. Advantageously,this method does not require measurement of the volume of biologicalsample.

In one aspect, the present invention is directed to a method forquantitation of glycated protein in a sample in which a solid supportmatrix which comprises a negatively charged group and a hydroxyborylcompound and which has a measurement area is contacted with an aliquotof the biological sample sufficient to cover the measurement area. Thesolid support matrix is then contacted with a first buffer which has apH selected so as to allow both glycated and non-glycated protein to bebound to the solid state matrix, in an amount sufficient to rinse offunbound protein. The amount of protein bound to the measurement area isquantitated by measurement of a selected property of the protein to givea first bound protein reading. The solid support matrix is thencontacted with a second buffer which has a pH selected to allow glycatedprotein to be bound to the solid support matrix but where non-glycatedprotein is not substantially bound to the solid support matrix, in anamount sufficient to rinse off unbound protein. The amount of proteinbound to the measurement area is quantitated by measurement of the sameselected property (as used to obtain the first bound protein reading) togive a second bound protein reading. The percentage of glycated proteinis calculated using the first and second bound protein readings.

According to an alternate aspect, the present invention is directed tomethods of quantitation of glycated protein in a sample in which a solidsupport matrix which comprises a negatively charged group and ahydroxyboryl compound and which has a measurement area is contacted withan aliquot of a biological sample sufficient to cover the measurementarea. The solid support matrix is then contacted with a first bufferwhich has a pH of about 5.0 to about 7.0 in an amount sufficient torinse off unbound protein. The amount of protein bound to themeasurement area is quantitated to give a first bound protein reading.The solid support matrix is then contacted with a second buffer whichhas a pH of about 8.0 to about 10.0 in an amount sufficient to rinse offunbound protein. The amount of protein bound to the measurement area isquantitated to give a second bound protein reading. The percentage ofglycated protein in the biological sample is calculated using the firstbound protein reading and the second bound protein reading.

The methods of the present invention may conveniently be used toquantitate amounts of either glycated hemoglobin or glycatednon-hemoglobin protein (such as albumin).

In the quantitation of glycated hemoglobin, typically a blood sample isused. The blood sample is contacted with a red blood cell lysing agentbefore the first bound protein reading is made. The sample may becontacted with the red blood cell lysing agent prior to being added tothe solid support (such as by pretreatment of the sample). Also, the redblood cell lysing agent may be added to the solid support by a pre-rinsebefore sample is added to the support. Alternatively, the first buffermay include a red blood cell lysing agent. Suitable red blood celllysing agents are known to those of skill in the art and include TritonX-100 and Igepal CA-630.

Preferably, the first and second bound protein readings involve anoptical reading. More preferably, the selected property measured isabsorbance or reflectance at a specified wavelength.

According to a preferred aspect of the present invention, thedihydroxyboryl compound of said solid support matrix has the structure;

wherein R is selected from the group consisting of phenyl, substitutedphenyl, hydrogen, and alkyl of 1 to about 6 carbon atoms. When R isalkyl, suitable alkyl groups include ethyl, 1-propyl, and3-methyl-1-butyl. Preferably R is m-amino-phenyl.

Suitable solid support matrices include supports selected from the groupconsisting of cellulose, nitrocellulose, cellulose acetate,polyacrylamide, agarose polyacrylaminde copolymer, agarose, starch,nylon, nylon polyesters, dextran, cross-linked dextran, dextranacrylamide copolymer, cross-linked hydroxyethylmethacrylate substitutedcross-linked polystyrenes, polyvinylalcohol, wool, metal oxides, porousceramics coated with hydrophilic organic polymers and glass. Preferablythe solid support matrix comprises a negatively charged group. Apreferred solid support matrix is cellulose. Preferably the negativelycharged group is selected from the group consisting of phosphate,sulfate, sulfonate, sulfinate and carboxylate (or carboxy). Preferablythe negatively charged group is carboxylate.

I The Assay

According to one aspect of the method of the present invention bothglycated and non-glycated protein are bound to a solid support matrixcomprising a negatively charged group and an immobilized dihydroxyborylcontaining compound under one pH condition. The bound non-glycatedprotein is then selectively removed under a second pH condition. Theglycated protein remains bound to the support under the second pHcondition. The measurement of total protein is made following thebinding under the first pH condition. The measurement of glycatedprotein is made after rinsing the support-bound protein complex underthe second pH condition to remove unbound protein. The percent glycationis calculated from the percent of the second measurement relative to thefirst. This assay is particularly useful for the measurement of glycatedhemoglobin and glycated protein in a blood, serum or plasma sample.

The method may be used for a biological sample such as capillary blood,whole blood, serum or plasma. According to a preferred aspect, onewavelength may be used for both measurements (total and glycated). Theassay is easily adaptable to be used with the same meters that are usedin self blood glucose monitoring by diabetics. Since both measurementsmay be made at the same location on the solid support (see Example 5),any differences in the support from location to location would notinterfere with the results.

II Solid Support Matrix and Immobilized Binder

The solid support matrix may be one of a number of natural or syntheticpolymeric materials to which can be bound negatively charged groups andthe dihydroxyboryl compound and through which the sample and reagentscan pass. Suitable support matrices include, for example, cellulose,nylon, nitrocellulose cellulose acetate, polyacrylamide, agarosepolyacrylamide copolymer agarose, starch, nylon polyesters, dextran,cross-linked dextran, dextran acrylamide copolymer, cross-linkedhydroxyethylmethacrylate substituted cross-linked polystyrenes andpolyvinyl alcohol. Other supports that may be utilized with the presentinvention include those listed in U.S. Pat. No. 4,269,605. In addition,other suitable supports are known to those of skill in the art. Apreferred solid support matrix is cellulose paper.

The dihydroxyboryl compound (preferably phenylboronic, boric or otherboronic acids, more preferably m-aminophenyboronic acid) may be bound tothe solid support by mechanical, physical or chemical means, preferablyby a covalent chemical bond. Binding to the solid support matrix can beby methods known in the art. Such methods include, for example, thoselisted in U.S. Pat. No. 4,269,605 to Dean et al, (issued May 26, 1989).

After binding the dihydroxyboryl compound to the support, the supportcan be rinsed with buffer or water. If the biological sample to beassayed has red blood cells, a detergent or other red blood cell lysingagent may optionally be included in the rinse if the assay is to measureglycated hemoglobin. Suitable detergents for use as a red blood celllysing agent include Triton X-100. The presence of lysing agent in thesupport may facilitate the lysing of red blood cells from a whole bloodsample. Alternatively, the sample may be pretreated with the red bloodcell lysing agent or the red blood cell lysing agent may be included inthe first buffer.

III Configuration of Solid Support Matrix

According to one aspect of the present invention, the solid supportmatrix is placed in a strip-type configuration along with othercomponents to handle the flow of sample and buffer. The sample andbuffers flow through the solid support matrix where binding occurs, andthen into an absorbent material which will soak up the excess solution,allowing adequate volumes of buffer to pass through. Flow of solutionthrough the strip can be along the length of the solid support matrix asin a lateral flow device, or through the thickness of the solid support,as in a vertical flow device (examples of such strip configurations aredepicted in FIGS. 6A, 6B and 6C).

Suitable materials for use in the solid support include, for example,cellulose (including cellulose paper), nitrocellulose, celluloseacetate, polyacrylamide. agarose polyacrylamide copolymer, agarose,starch, nylon, nylon polyester, dextran, cross-linked dextran, dextranacrylamide copolymer, cross-linked hydroxyethylmethacrylate substitutedcross-linked polystyrenes, polyvinylalcohol, wool, metal oxides, porousceramics coated with hydrophilic organic polymers and glass.

IV The Sample and Preferred Buffers

Suitable biological samples for use according to the methods of thepresent invention include whole blood, serum and plasma. Red blood cellscan be removed from the sample if desired by use of methods described inthe art. Included are such methods using glass fibers (U.S. Pat. No.4,477,575 issued Oct. 16, 1984 to Vogel et al.), carrier containingcarbohydrate (U.S. Pat. No. 4,678,757 issued Jul. 7, 1987 to Rapkin) ora matrix containing a polyol (U.S. Pat. No. 5,725,774 issued Mar. 10,1998 to Neyer).

According to a preferred aspect (especially for quantitation ofnon-hemoglobin glycated protein), the sample can be pretreated with aprotein specific binding agent such as “dye” to label proteins and whichmay facilitate measurement of their concentration. A variety of suchdyes known to those skilled in the art may be utilized with the methodof the present invention. For example, a fluorescent dye such asfluorescein isothiocyanate (“FITC”) may be used.

The sample is added to the solid support and allowed to flow through thesupport. The flow can be through the length of the support or throughthe thickness. The volume of sample added can range from a volume justlarge enough to cover the measurement area uniformly (such as about 3 to5 μL) to a volume that will pass through the support in a reasonabletime (approximately 40 μL). The actual volume limits will depend on thedimensions of the solid support and the other strip components. A larger(wider or longer) strip will be able to handle larger volumes of sample,but will require larger minimum volumes to cover the measurement area. Asmaller strip will function with small volumes, but will not be able tohandle large volumes.

After the sample has been absorbed into the strip, the first buffer isadded. The pH of the first buffer is preferably between about 5.0 andabout 7.0. More preferably, the pH of the first buffer is about 5.5 toabout 7.0 for quantitation of glycated hemoglobin and about 5.0 to about6.5 for quantitation of glycated albumin. The buffer contains a suitablebuffering agent which has a pKa appropriate for control of the pH withinthe given range, but that does not otherwise affect the binding of theprotein. Buffering agents suitable for use in the first buffer are knownto those of skill in the art. Preferred buffering agents for use in thefirst buffer include MES, MOPS and HEPES. The buffering agent is presentin a concentration sufficient to maintain the pH in the desired range.Suitable concentrations for the buffering agent may range from about 5mM to about 500 mM. If the biological sample includes red blood cells,the buffer may further comprise a cell lysing agent such as, forexample, Triton X-100 or Igepal CA-630. The buffer volume added can bestandardized to a specific number of drops adequate to rinse throughexcess, non-bound sample. The adequacy of rinsing can also be monitoredby the reflectance meter taking multiple measurements with the finalmeasurement taken when the change is essentially zero.

The second buffer preferably has a pH from about 8.0 to about 10.0. Thesecond buffer contains a suitable buffering agent which has a pKappropriate for control of pH within the given range, but that does notinterfere with the binding of glycated protein to the immobilizeddihydroxyboronate compound. Buffering agents suitable for use in thesecond buffer are known to those of skill in the art. Preferredbuffering agents for use in the second buffer include ammonium acetateand taurine. The buffering agent is present in a concentrationsufficient to maintain the pH in the desired range. Suitableconcentrations for the buffering agent may range from about 5 mM toabout 500 mM, or, alternatively, up to the solubility limit of thebuffering agent. The adequacy of rinsing can be monitored as describedfor the first buffer. Optionally, the buffer can contain a divalentmetal ion such as Mg⁺⁺ to help stabilize the binding of the protein tothe boronate.

V Quantitation

The amount of protein bound to the support after each rinse can bequantitated using measurement methods known to those skilled in the artincluding measurement of optical absorbance or reflectance at anappropriate wavelength.

According to a preferred aspect of the method, a first reflectancemeasurement is made of the solid support after sample is added and theexcess is rinsed through using the first buffer. This reflectancemeasurement is used in calculating the total protein concentration usingan algorithm derived from calibration data or using “K/S” values asvalues proportional to concentration. A second reflectance measurementis made after washing off the non-glycated protein from the solidsupport using the second buffer. This measurement is used to calculatethe glycated protein concentration in the same way as the firstmeasurement. The percent glycated protein is calculated as the ratio ofthe glycated protein concentration to the total proteinconcentration×100.

When light enters a transparent media, some of it is absorbed and somepasses through the media. The Beer-Lambert law provides that the amountof light absorbed (“A”) is the product of the concentration of thematerial absorbing the light (“C”) and the length the light travelsthrough the media (“d”) (i.e. A=ecd, wherein e is the extinctioncoefficient of the absorbing material). When light enters a solid, theamount that reflects off the solid is determined by what is absorbed bythe solid and what is scattered by the solid. The model used to definethis relationship is the Kubelka-Munk Theory. This model defines twodifferential equations for the change in the light intensity as itpasses into the solid and as it is scattered back out of the solid. Theequations include the symbol “K” which is the absorption coefficient insolid phase and the symbol “S” which is the scattering coefficient. Inthe simplified derivation, the equations reduce to the following:K/S=[(1−R)^2]/(2R) where R is the measured reflectance and K/S isproportional to the concentration of the absorbing/scattering material.In certain of the Figures (see, e.g., FIGS. 7 and 8) the data takenusing the reflectance meter are plotted with y values of K/S which areproportional to the concentration of the analyte.

The methodology used in the assay is adaptable for use in a strip thatcan be used with hand held reflectance meters used in glucose assays.

The total amount of protein that binds to the solid support iscontrolled by the binding capacity of the negatively charged groupsimmobilized on the support. We have observed that glycated andnon-glycated protein rapidly bind to the derivatized support in the sameratio as in the sample. The volume of sample added to the support doesnot have to be measured since any excess will be rinsed through.

The reported percent glycation is calculated from the ratio of twomeasurements made at the same location on the solid support in thestrip. Therefore, variations in the total amount of protein bound bydifferent strips will not affect the reported result. Since both totaland glycated protein are measured using the same method at the samelocation on the solid support, variations which could affect themeasurements will have the same effect on both measurements, minimizing“noise”.

To assist in understanding, the present invention will now be furtherillustrated by the following examples. These examples, as they relatedto the present invention, should not, of course, be construed asspecifically limiting the invention. Also, such variations of theinvention, now known or later developed, which would be within thepurview of one skilled in the art are considered to fall within thescope of the present invention, as described herein and herein afterclaimed.

EXAMPLES Example 1 Preparation of the Boronated Solid Support Matrix

According to a preferred aspect of the detection method of the presentinvention, a solid support matrix suitable for use in a stripconfiguration is prepared by covalently attaching a derivative ofboronate, m-aminophenylboronate to a carboxy cellulose based materialusing a conventional linking chemistry.

Materials

-   1. Solid Support Matrix: cellulose based solid with carboxylic acid    groups covalently bound (Sartobind C membrane, Sartorious).-   2. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)    (Pierce)-   3. Boronate derivative: m-aminophenylboronic acid (Sigma-Aldrich)-   4. Buffer: 0.1M MES buffer, pH 6.5    Procedure

A 46.7 mg portion of m-aminophenylboronate is dissolved in 25 mL of 100mM MES buffer. The pH is re-adjusted to 6.5 after the boronatedissolves. A 28.9 mg portion of EDC is dissolved in the MES-boronatesolution. The solid support matrix is soaked in the solution for thedesired time (25 mL of solution was enough to treat a 30 cm² piece ofmatrix). The matrix is removed from the solution and rinsed in the MESbuffer and allowed to air dry.

The binding characteristics of the boronated solid support matrixprepared as above using different soak times were measured using theelution assay described in Example 2. The resulting binding of total(open triangles) and glycated hemoglobin (closed triangles) is depictedin FIG. 1. Open circles show the ratio of glycated to total hemoglobin.

As more boronate groups are added to the solid support matrix, fewercarboxylic groups are left for ionic binding. The ratio of glycatedhemoglobin to total hemoglobin increases until there is a sufficiency ofboronate groups, then the ratio remains the same even through the totalbinding is decreased.

Example 2 Effect of Variable Incubation Times

An assay using the boronated support of Example 1 in a singlemeasurement assay (“Single Measurement Method”) was compared to a methodof the present invention to determine the effect of variable incubationtimes on the concentration results obtained with each method.

The following assays were performed to compare the two methods. In theSingle Measurement Method assay, blood lysate samples from anon-diabetic individual and a diabetic individual were diluted 1:1 with500 mM ammonium acetate buffer pH=9.5 with 50mM Mg₊₊ and the sampleswere allowed to be in contact with the boronated solid support matrixprepared as in Example 1 for a variety of time periods. Followingincubation, the boronated solid supports were rinsed with ammoniumacetate buffer pH=9.5. Glycated hemoglobin was eluted with an elutionbuffer comprised of tris buffer at pH 8.0 containing 200mM sorbitol. Theabsorbance of the eluent was measured at 415 mM.

In the second assay, corresponding to the method of the presentinvention, blood lysate samples from a non-diabetic individual and adiabetic individual were diluted 1:1 with mM MES buffer pH=6.5. Thesamples were allowed to be in contact with the boronated solid supportmatrix for time periods identical to those used for the first assay andwere incubated at a similar time periods as done with the first assay.Following incubation the diluted samples were discarded and theboronated solid supports were rinsed with MES buffer pH=6.5. The solidsupport matrixes were then rinsed a second time with 500 mM ammoniumacetate buffer pH=9.5 containing 50 mM Mg⁺⁺. The absorbance of the rinsewas measured at 415 nm corresponding to non-glycated hemoglbin. Glycatedhemoglobin was eluted off the matrix and the absorbance of this rinsewas measured. The data obtained from the diabetic sample is provided inthe Table I (see also FIGS. 2 and 3).

TABLE I Diabetic blood lysate sample Single dwell Present InventionMeasurement time (min) total gly Gly/total Gly 0.25 0.769 0.12 0.1560.029 0.5 0.739 0.109 0.147 0.032 1 0.824 0.118 0.143 0.037 2 0.8460.116 0.137 0.038 4 0.838 0.123 0.147 0.047 8 0.856 0.126 0.147 0.072 160.814 0.125 0.154 0.082 4 0.779 0.118 0.151 0.049 8 0.844 0.103 0.1220.064 16 0.857 0.121 0.141 0.082

Linear regression analysis of this data clearly showed, that the resultsobtained using the single measurement method were heavily dependent onincubation time. However, when using the methods of the presentinvention the results obtained were independent of incubation time,which it is believed will provide more reliable results and make theassay more robust and suitable for untrained users.

Example 3 Effect of pH on Binding

The pH of the buffer affects the amount of hemoglobin binding to theboronated support. If the pH is low, then both glycated and non-glycatedhemoglobin are bound. If the pH is high, only glycated hemoglobin willbind. FIG. 4 depicts the binding of hemoglobin to the carboxy cellulosemembrane (no boronate groups present) following the assay proceduredescribed in Example 2. The membrane loses its ionic binding ofhemoglobin between pH 6 and 7 when boronate is not present.

FIG. 5 depicts the hemoglobin and human serum albumin binding propertiesof the carboxy cellulose support with added boronate groups, prepared asdescribed in Example 1. Binding of the protein occurs at lower pH's dueto the negatively charged carboxyl groups. The protein bound at thehigher pH, due to the phenylboronate, is the glycated protein present inthe samples.

Example 4 Description of Exemplary Strip Design Configurations

The boronated solid support matrices containing negatively chargedgroups can be incorporated in any assay where the separation of glycatedfrom non-glycated protein is required. According to the preferred assaymethods of the present invention the boronated solid support matrix isused in combination with other components to direct the flow of thesample and buffers. These components may be in the form of a strip whichcan be placed into a small, hand held reflectance meter forquantitation. Suitable alternative configurations for the strip aredepicted in FIGS. 6A, 6B and 6C.

In the lateral flow strip configuration (FIG. 6A), the fluids movethrough the length of the boronated support (parallel to the surface ofthe support). In the vertical flow (FIG. 6B) and combination stripconfiguration

(FIG. 6C), the movement of fluid is more through the thickness of thesolid support matrix (perpendicular to the surface).

Each configuration depicted has a sample application site (1, 11 or 21)which is a hole in a piece of plastic (normally white for appearances),(4, 14 or 24). The boronated solid support (2, 12 or 22) is in contactwith wicking material (6, 16 or 26). In the configuration depicted inFIG. 6A, fluids flow from the application area (1) through the wickingmaterial (6) to the support (2) and through the support to the reservoir(3). In the configurations depicted in FIGS. 6B and 6C, fluids flow fromthe application area (11 or 21) and boronated support (12 or 22) to thewicking material (16 or 26) and the reservoir (13 or 23). All thecomponents are placed on top of the bottom piece of plastic, eitherwhite (15) or clear (5 or 25) depending on the configuration. Thereflectance of the boronated support is measured by shining a light ofthe selected wavelength onto the support surface and measuring itsintensity (7, 17 or 27). In the configurations depicted in FIGS. 6B and6C, there is a piece of white reflecting material (18 or 28) placedbehind the support to reflect light passing through the support backthrough it to reduce loss of intensity.

Example 5 Assay Proportionality: Glycated Hemoglobin

A vertical flow strip design described in Example 4 and as depicted inFIG. 6D was used in assays of mixtures of blood lysates from normal anddiabetic blood samples. Approximately 8 μl of blood lysate was depositedon the boronated solid support matrix prepared as in Example 1. Theapplication site was then rinsed with ˜50 μL MES buffer pH=6.5 (100 mM)and a reflectance measurement at 430 nm was recorded. The applicationsite was then rinsed with ˜50 μL ammonium acetate buffer (500 mM)pH=9.5, containing 50 mM Mg⁺⁺ and a second reflectance measurement at430 nm was recorded at the application site. The reflectance values wereconverted to K/S values which are proportional to the concentration ofhemoglobin being measured. The data are shown in Table II below (seeFIGS. 7 and 8).

TABLE II Reflectance k/s norm.:diab. % gly hb* Total gly Total gly ratio(gly/total) 1:0 4.5 0.186 0.428 1.781 0.382 0.215 4:1 6.1 0.219 0.4481.393 0.340 0.244 3:2 7.7 0.197 0.415 1.637 0.412 0.252 2:3 9.3 0.1650.350 2.113 0.604 0.286 1:4 10.9 0.197 0.395 1.637 0.463 0.283 0:1 12.50.179 0.363 1.883 0.559 0.297

The regression statistics of the data plots are shown in the Table IIIbelow.

TABLE III plot Slope intercept S_(y.x) CV* Total Hb 0.031 1.48 0.25514.7 Glycated Hb 0.026 0.24 0.076 15.9 Gly/total 0.010 0.18 0.010 3.8*calculated using the mean values for each fraction

The plots, S_(y,x) and resulting C.V.'s show that the total and glycatedvalues measured vary from strip to strip. However, the large variationsare reduced when the ratio of the two values is taken, demonstrating theadvantage of using the methods of the present invention.

The results show that the calculated glycated fraction is proportionalto the percent glycated hemoglobin in the sample.

The term CV is the coefficient of variation and is the measure of thescatter of replicate measurements around a mean value. The coefficientis calculated from the standard deviation (“SD”) and the mean value(“M”) of the replicates (i.e. CV=100×SD/M). The benefit to using thisvalue is that because it is expressed as a percentage it may be comparedto other CV values without the requirement of knowing the mean values.

The term S_(y.x) is a measure of variability used in linear regressioncalculation. It is the measure of the variability of “y” after removingthe effect of “X” (i.e. it measures the variability of the data aroundthe regression line).

Example 6 Assay Proportionality: Glycated Protein

A normal albumin sample was obtained from serum from a non-diabeticindividual. An elevated glycated albumin sample was prepared from humanserum albumin glycated in vitro (see, U.S. Pat. No. 5,589,393 to MichaelD. Fiechtner, et al., issued Dec. 31, 1996).

Both samples were assayed for fructosamine amount using a manualfructosamine assay based on Tietz, Textbook of Clinical Chemistry, 2ndEd. (W. B. Saunders Co., Carl A. Burtis, Edward Ashwood, Eds., 1994),page 986–988. The elevated glycated albumin sample was found to contain5.5 times the concentration of fructosamine as the normal albuminsample. The samples were mixed together in the proportions shown inTable IV (see also, FIGS. 9 and 10).

The resulting samples were assayed in accordance with the presentinvention as described in Example 2 and the A280 absorbance valuesrecorded in Table IV.

TABLE IV Relative Fructosamine measured Measured calculated Conc.total-gly glycated total gly/total 1.0 0.065 0.027 0.092 0.029 1.0 0.0670.028 0.095 0.029 2.1 0.056 0.037 0.093 0.040 2.1 0.052 0.037 0.0890.042 3.3 0.045 0.058 0.103 0.056 3.3 0.049 0.045 0.094 0.048 4.4 0.0400.053 0.093 0.057 4.4 0.038 0.054 0.092 0.059 5.5 0.033 0.063 0.0960.066 5.5 0.030 0.072 0.102 0.071

Regression statistics for the above plot are given in the Table V below.

TABLE V Fraction slope Intercept R{circumflex over ( )}2 total 0.0010.091 0.177 glycated 0.009 0.020 0.908 gly/total 0.008 0.022 0.962

The calculated glycated albumin fraction was observed to be proportionalto the fructosamine content. The results are proportional from the lowsample concentration to 5.5 times the low concentration as shown by theregression statistics of R^2. Therefore the assay method used to measureglycated hemoglobin is also suitable for the measurement of glycatedalbumin.

Example 7 Effect of Sample Volume

A lateral flow strip format as described in Example 4 and depicted inFIG. 6A was used to assay non-diabetic and diabetic whole blood atvaried sample volumes to determine the effect of sample volume on theassay, which followed the method described in Example 2. The measuredabsorbance values (415 nm) are presented in the Table VI (see FIGS. 11to 12).

TABLE VI absorbance (415) μL sample total Hb Gly Hb gly/total 10 1.1640.183 0.157 20 1.053 0.167 0.159 30 1.233 0.164 0.133 40 1.206 0.1950.162

The regression statistics are shown in the Table VII.

TABLE VII Fraction slope Intercept S_(y.x) slope = 0? Total Hb 0.00310.088 0.084 yes (P = 0.502) gly Hb 0.0003 0.169 0.017 yes (P = 0.706)Gly/total −0.0001 0.156 0.016 yes (P = 0.893)

The results demonstrate that binding of total hemoglobin and glycatedhemoglobin in the assay are independent of sample volume when the volumeranges from about 10 μL to about 40 μL in assays of whole blood.

Example 8 Effect of Variation of Total Hemoglobin Concentration

The effect of varying the amount of total hemoglobin in the assay wasdetermined using the assay procedure described in Example 2 to assaydiluted blood samples. Table VIII below list the absorbance values (415nm) from the assays of non-diabetic and diabetic blood lysates dilutedby factors of 2, 4, 8 and 16 (see FIGS. 13 to 15).

TABLE VIII dil A (415 nm) Sample factor Total Hb gly % gly Normal 21.309 0.091 6.9 4 0.754 0.054 7.2 8 0.382 0.026 6.8 16 0.178 0.014 7.9Diabetic 2 1.561 0.275 17.6 4 0.929 0.157 16.9 8 0.467 0.084 18.0 160.199 0.036 18.1

The regression statistics are listed in the Table IX below for thegraphs.

TABLE IX sample slope Intercept S_(y.x) slope = 0? normal 0.00060 0.0670.0035 yes (P = 0.202) Diabetic 0.00058 0.172 0.0049 yes (P = 0.329)

The amounts to total hemoglobin and glycated hemoglobin that bind to thesolid support matrix decrease with decreasing total hemoglobinconcentration. The calculated glycated hemoglobin/total hemoglobin,however, was constant over the dilutions assayed as seen in theregression statistics (i.e. slopes are essentially zero). Thisdemonstrated the advantage of the methods of the present invention (i.e.using the ratio of two measurements to produce results independent ofsample dilution or hemoglobin concentration).

Example 9 Comparison to Other Commercial Assays

Twenty-five whole blood samples were assayed using the stripconfiguration depicted in FIG. 6C, and described in Example 4 andfollowing the assay method described in Example 5. The samples were alsoassayed using two commercially available tests for hemogoblin A1c.

The commercially available tests compared to the method of the presentinvention were:

1. Roche Integra 400 (Roche Diagnostics)

The assay was performed by a desktop, multi-sample analyzer using animmunoturbidimetric determination of the glycated N-terminal valine ofthe β-chain in hemoglobin or HbA1c according to the manufacturer'sinstructions. Each sample was assayed once by this method.

2. DCA 2000 (Bayer)

The assay was performed by a desktop, single assay analyzer using animmunoturbidimetric determination of HbA1c according to themanufacturer's instructions. Each sample was assayed once using thisassay.

The results are plotted in FIGS. 16A and 16B and show that the resultsobtained using the compared methods and the methods of the presentinvention correlate.

1. A method for determining a ratio of an amount of a glycated form of aprotein to a total amount of the protein in a sample containing bothglycated and nonglycated forms of the protein, comprising: providing asolid support having negatively charged carboxyl groups immobilizedthereon, which groups are capable of binding both the glycated and thenonglycated forms of the protein at a first pH, said support also havinghydroxyboryl groups immobilized thereon, interspersed with thenegatively charged carboxyl groups, which hydroxyboryl groups arecapable of binding the glycated form of the protein at a second pH;adding the sample to the solid support at the first pH, thereby bindingboth the glycated and the nonglycated forms of the protein to thenegatively charged carboxyl groups on the solid support, and thenperforming a first measurement indicative of the total amount of theglycated and the nonglycated forms of the protein bound to the solidsupport; changing the pH on the support to the second pH, therebyremoving both the nonglycated form of the protein and the glycated formof the protein from the negatively charged carboxyl groups, after whichremoval the glycated form of the protein immediately binds to thehydroxyboryl groups on the support independent of incubation time, andthen performing a second measurement indicative of the amount of theglycated form of the protein bound to the solid support; and determiningthe ratio of the amount of the glycated form of the protein to the totalamount of the glycated and the nonglycated forms of the protein in thesample from the first and second measurements.
 2. The method of claim 1,wherein the first pH is achieved by applying a buffer of about pH 5.0 to7.0.
 3. The method of claim 1, wherein the second pH is achieved byapplying a buffer of about pH 8.0 to 10.0.
 4. The method of claim 1,wherein the glycated protein is hemoglobin.
 5. The method of claim 1,wherein the glycated protein is albumin.
 6. The method of claim 1,wherein the sample comprises blood.
 7. The method of claim 1, whereinthe sample comprises serum.
 8. The method of claim 1, wherein the samplecomprises plasma.
 9. The method of claim 1, wherein the first and secondmeasurements measure an optical property of the protein.
 10. The methodof claim 1, wherein the first and second measurements are opticalreadings at a predetermined wavelength.
 11. The method of claim 1,wherein the first and second measurements measure a protein label. 12.The method of claim 1, wherein the hydroxyboryl group is of the type

where R is selected from the group consisting of phenyl, alkyl of 1–6carbons, ethyl, 1-propyl, 3-methyl-1-butyl and aminophenyl.
 13. Themethod of claim 1, wherein the solid support is selected from the groupconsisting of cellulose, nitrocellulose, cellulose acetate,polyacrylamide, agarose polyacrylamide copolymer, agarose, starch,nylon, nylon polyesters, dextran, cross-linked dextran, dextranacrylamide copolymer, cross-linked hydroxyethylmethacrylate, substitutedcross-linked polystyrenes, polyvinylalcohol, wool, metal oxides, porousceramics coated with hydrophilic organic polymers and glass.
 14. Amethod for determining a ratio of an amount of glycated albumin to atotal amount of glycated and nonglycated albumin in a sample containingboth glycated and nonglycated albumin, comprising: providing astrip-type device comprising: (1) a solid support matrix having ameasurement area; and (2) negatively charged carboxyl groups anddihydroxyboryl groups immobilized and interspersed on the solid supportmatrix, said negatively charged carboxyl groups are capable of bindingboth glycated and nonglycated albumin at a first pH between about 5.0and about 7.0, and said dihydroxyboryl groups are capable of bindingglycated albumin at a second pH between about 8.0 and about 10.0; addingthe sample to the solid support matrix at the first pH, thereby bindingboth glycated and nonglycated albumin to the negatively charged carboxylgroups on the solid support matrix, and then performing a first opticalmeasurement on the measurement area indicative of the total amount ofglycated and nonglycated albumin bound to the solid support matrix;changing the pH on the solid support matrix to the second pH, therebyremoving both the nonglycated albumin and the glycated albumin from thenegatively charged carboxyl groups, after which removal the glycatedalbumin immediately binds to the dihydroxyboryl groups on the solidsupport matrix independent of incubation time, and then performing asecond optical measurement on the measurement area indicative of theamount of glycated albumin bound to the solid support matrix; anddetermining the ratio of the amount of glycated albumin to the totalamount of glycated and nonglycated albumin in the sample from the firstand second optical measurements.
 15. The method of claim 14, whereinsaid dihydroxyboryl groups have the formula:

where R is selected from the group consisting of phenyl, alkyl of 1–6carbons, ethyl, 1-propyl, 3-methyl-1-butyl and aminophenyl.
 16. Themethod of claim 14, wherein the solid support matrix is selected fromthe group consisting of cellulose, nitrocellulose, cellulose acetate,polyacrylamide, agarose polyacrylamide copolymer, agarose, starch,nylon, nylon polyesters, dextran, cross-linked dextran, dextranacrylamide copolymer, cross-linked hydroxyethylmethacrylate, substitutedcross-linked polystyrenes, polyvinylalcohol, wool, metal oxides, porousceramics coated with hydrophilic organic polymers and glass.
 17. Themethod of claim 16, wherein said solid support matrix is carboxycellulose.
 18. The method of claim 14, wherein said first pH is achievedwith a buffer selected from the group consisting of MES, MOPS and HEPES.19. The method of claim 14, wherein said second pH is achieved with abuffer selected from the group consisting of ammonium acetate ortaurine.
 20. The method of claim 14, wherein the sample comprises blood,plasma or serum.
 21. A method for determining a ratio of an amount ofglycated hemoglobin to a total amount of glycated and nonglycatedhemoglobin in a sample containing both glycated and nonglycatedhemoglobin, comprising: providing a strip-type device comprising: (1) asolid support matrix having a measurement area; and (2) negativelycharged carboxyl groups and dihydroxyboryl groups immobilized andinterspersed on the solid support matrix, said negatively chargedcarboxyl groups are capable of binding both glycated and nonglycatedhemoglobin at a first pH between about 5.0 and about 7.0, and saiddihydroxyboryl groups are capable of binding glycated hemoglobin at asecond pH between about 8.0 and about 10.0; adding the sample to thesolid support matrix at the first pH, thereby binding both glycated andnonglycated hemoglobin to the negatively charged carboxyl groups on thesolid support matrix, and then performing a first optical measurement onthe measurement area indicative of the total amount of glycated andnonglycated and nonglycated hemoglobin bound to the solid supportmatrix; changing the pH on the solid support matrix to the second pH,thereby removing both the nonglycated hemoglobin and the glycatedhemoglobin from the negatively charged carboxyl groups, after whichremoval the glycated hemoglobin immediately binds to the dihydroxyborylgroups on the solid support matrix independent of incubation time, andthen performing a second optical measurement on the measurement areaindicative of the amount of glycated homoglobin bound to the solidsupport matrix; and determining the ratio of the amount of glycatedhemoglobin to the total amount of glycated and nonglycated hemoglobin inthe sample from the first and second optical measurements.
 22. Themethod of claim 21, wherein said dihydroxyboryl groups have the formula:

where R is selected from the group consisting of phenyl, alkyl of 1–6carbons, ethyl, 1-propyl, 3-methyl-1-butyl and aminophenyl.
 23. Themethod of claim 21, wherein the solid support matrix is selected fromthe group consisting of cellulose, nitrocellulose, cellulose acetate,polyacrylamide, agarose polyacrylamide copolymer, agarose, starch,nylon, nylon polyesters, dextran, cross-linked dextran, dextranacrylamide copolymer, cross-linked hydroxyethylmethacrylate, substitutedcross-linked polystyrenes, polyvinylalcohol, wool, metal oxides, porousceramics coated with hydrophilic organic polymers and glass.
 24. Themethod of claim 21, wherein said solid support matrix is carboxycellulose.
 25. The method of claim 21, wherein said first pH is achievedwith a buffer selected from the group consisting of MES, MOPS and HEPES.26. The method of claim 21, wherein said second pH is achieved with abuffer selected from the group consisting of ammonium acetate ortaurine.
 27. The method of claim 21, wherein the sample comprises blood,plasma or serum.